Novel chromogenic thrombin substrates

ABSTRACT

Substrate with high specificity to thrombin and thrombin-like enzymes having the formula: ##STR1## and salts thereof, wherein R 1  is H or OH; R 2  is nitrophenyl; and n is 1, 2 or 3 provided along with the method of use.

The present invention relates to novel chromogenic substrates forthrombin and thrombin-like enzymes. The substrates according to theinvention are especially suitable for a quantitative determination ofthrombin or for a study of reactions in which thrombin is formed,inhibited or consumed, or for determination of factors which exert aninfluence or take part in such reactions, e.g. for determination ofanti-thrombin, prothrombin and heparin.

Synthetic substrates for enzyme determinations have great advantages ascompared to the natural ones, provided that they fulfill certainconditions, such as a great sensitivity for and specificity for theenzyme, a good solubility in water or the biological test liquid andeasy detectability of some of the splitting products.

One of the hitherto best substrates for thrombin determination isdescribed in our Swedish Pat. No. 380.257 and consists of thechromogenic tripeptide derivative (as regards the abbreviations cf. page4):

    Bz--Phe--Val--Arg--pNA (S--2160)                           A

This has a great sensitivity for thrombin and gives upon enzymatichydrolysis the chromophoric product p-nitroaniline which easily can bedetermined spectrophotometrically. S-2160 has, however, a delimitationdue to its relatively low solubility (1 mg/ml). A low solubility causesthe disadvantage that one has to work very near the saturation limit forthe substrate so as to achieve a satisfactory substrate concentration.In enzyme determination in different biological systems a precipitationof the substrate as such can occur or a combined protein/substrateprecipitation. The said precipitations will cause erroneousspectrophotometer readings and thus erroneous enzyme determinations. Theenzyme substrate S-2160 becomes considerably more soluble if the benzoylgroup is replaced with H, thus:

    H--Phe--Val--Arg--pNA                                      B

the now free protonized amino group on Phe increases the solubility, butcauses also that the velocity with which thrombin splits the substratedecreases heavily, about 30 times (cf. Table I). Further, the substratecan, in a biological test solution, in a non-desired way be decomposedfrom the N-terminal end by amino peptidases.

According to the present invention the substrate according to formula Bhas been modified by exchanging Val for a cyclic imino acid (Aze, Pro orPip) and L-Phe with D-Phe. As expected the substrates so obtained arestill very soluble but quite surprisingly the activity against thrombinis not decreased but instead it is 30-50 times higher than the activityfor the corresponding substrate with solely L-amino acids (Table I).Further, the novel substrates are about 400% more active than S-2160.The N-terminal D-amino acid also prevents a non-desired attack by aminopeptidases since the latter are specific for L-amino acids. The novelchromogenic substrates according to the invention are characterized bythe following general formula: ##STR2## or salts thereof, wherein R₁ canbe hydrogen or hydroxy. R₂ can be chosen among nitrophenyl, naphthyl,nitronaphthyl, methoxynaphthyl, quinolyl or nitroquinolyl, and n can be1, 2 or 3.

For the synthesis of the novel chromogenic enzyme substratesconventional protective groups and coupling methods are used all ofwhich are well-known within the peptide chemistry.

As an α-amino protective group carboxybenzoxy or t-butyloxycarbonylgroups can be used with advantage or any group related thereto such asfor instance p-methoxy, p-nitro or p-methoxyphenylazo carbobenzoxy.

As a protection for the δ-guanido group of the arginyl group it isadvantageous to use protonization, an NO₂ -group or ap-toluene-sulphonyl group.

As protection for the hydroxy group in the tyrosine group it is ofadvantage to use a t-butyl or benzyl group. As a splittable carboxyprotective group it is suitable to use methyl, ethyl or benzyl ester.

The coupling between two amino acids or a dipeptide and an amino acid isachieved by activation of the α-carboxy group. The activated derivativecan either by isolated or generated in situ and can be for instancep-nitrophenyl, trichlorophenyl, pentachlorophenyl orN-hydroxysuccinimide ester, symmetric or asymmetric anhydride, acidazid, or N-hydroxybenzotriazole ester.

The principle for the synthesis can be a stepwise coupling of the aminoacids to the C-terminal arginyl group, either already provided with acoupled chromophoric group which functions as a carboxy protective groupor provided with a splittable carboxy protective group, and thechromophoric group is then coupled to the protected tripeptidederivative, or alternatively it is possible to synthesize the N-terminaldipeptide fragment per se which then is coupled to the arginyl groupwith or without a chromophoric group according to the statements above.

Independently of the chosen principle a purification of intermediary andend products by gel filtration chromatography is suitable since thiswill enable a rapid synthesis work and gives maximal yields.

TLC analysis has been made partly of eluates from GPC and partly ofevaporated and dried end and intermediary products.

The invention is described in more detail in the following non-limitingspecific examples.

    ______________________________________                                        Abbreviations                                                                 Amino acids (if not otherwise stated the L-form is meant):                    ______________________________________                                        Arg    = Arginine                                                             Aze    = 2-Azetidine carboxylic acid                                          Phe    = Phenylalanine                                                        Pip    = Pipecolinic acid                                                     Pro    = Proline                                                              Val    = Valine                                                               AcOH   = Acetic acid                                                          Bz     = Benzoyl                                                              Cbo-   = Carbobenzoxy-                                                        DMF    = Dimethylformamide                                                    Et.sub.3 N                                                                           = Triethylamine                                                        EtOAc  = Ethylacetate                                                         HMPTA  = N, N, N', N', N", N"-hexamethylphosphoric                                    acid triamide                                                         GPC    = Gel filtration chromatography                                        MeOH   = Methanol                                                             OpNP   = p-nitrophenoxy                                                       pNA    = p-nitroanilide                                                       TLC    = Thin-layer chromatography                                            ______________________________________                                    

Thin-layer chromatography:

For TLC-analysis preprepared glass plates are used with silica gel F₂₅₄(Merck) as absorption agent. The solvent systems used are the following:

P₁ : chloroform: MeOH 9:1 (volume ratio)

A : n-butanol : AcOH : water 3:2:1 (volume ratio)

After finished chromatography the plate is studied in UV-light (254 nm)and development is made subsequently with chlorine/o-toluidine reagentaccording to common practice. When a "homogenous product according toTLC" is stated the analysis is performed on an amount of μg. The R_(f)values stated are results from separate chromatographic procedures.

The gel Sephadex® G-15 used for the gel filtration is a crosslinkeddextran gel. The gel Sephadex® LH-20 is a hydroxypropylated crosslinkeddextran gel. The ion exchanger Sephadex® QAE-25 used is a crosslinkeddextran gel with diethyl-(2-hydroxy-propyl)-amino-ethyl as functionalgroup. These gels are from Pharmacia Fine Chemicals, Uppsala, Sweden.

Description of the synthesis

I. Cbo--Arg(NO₂)pNA

35.3 g (10 mmol) of dry Cbo--Arg(NO₂)--OH are dissolved in 200 ml of dryfreshly distilled HMPTA at room temperature, whereupon 10.1 g (100 mmol)of Et₃ N and 24.6 g (150 mmol) p-nitrophenyl isocyanate is added duringstirring and moisture-free conditions. After a reaction time of 24 h thesolution is poured down in 2l of 2% sodiumbicarbonate solution understirring. The precipitation formed is removed by filtration and washedwith 2% bicarbonate solution, water, 0.5 N HCl (aq) and water andfinally dried. The raw product is extracted with warm MeOH anddifficultly soluble by-products are filtrated. The filtrate is purifiedby chromatography on a column of Sephadex® LH-20, swelled in and eluatedwith MeOH. Yield: 29.8 g (63%) Analysis: Homogenous according to TLC inP₁ (R_(f) : 0.34) [α]_(D) ²⁴ + 20.5° (c 1.9, DMF)

II. Cbo--Pro--Arg(NO₂)-pNA

4.8 g (10 mmol) of Cbo--Arg(NO₂)--pNA are slurried in 25 ml of dry AcOH,whereupon 15 ml of 5.6 N HBr in AcOH are added. After a reaction periodof 50 min at room temperature the solution is poured under vigorousstirring into 300 ml of dry ether. The ether phase is sucked from theprecipitation obtained and the precipitation is washed with 2 portionsof 100 ml of ether each. The HBr.H--Arg(NO₂)--pNA thus obtained is driedin vacuum over NaOH at 40° C for 16 h. It is subsequently dissolved in25 ml of DMF and the solution is cooled to +10° C. Now Et₃ N is added inan amount sufficient for giving a moist pH-paper kept immediately abovethe surface of the solution a weakly basic reaction (1.9 ml).Precipitated Et₃ N.HBr is removed by filtration and 4.1 g (11 mmol) ofCbo-Pro-OpNP are added. After 1 h further 0.7 ml Et₃ N are added andalso after 4 h. The solution is allowed to adjust to room temperatureover the night. So as to avoid that the excess of Cbo-Pro-OpNPcontaminates the end product during GPC since they have similar eluationvolumes in the chromatographic system used 0.5 ml (5 mmol) of n-butylamine is added. After 30 min 10 mmol of diluted HCl are added, thereaction solution is evaporated on a rotavapor, stirred with a couple ofportions of water which is removed by decantation. The residue isdissolved in MeOH and chromatographed on a column of Sephadex.sup.(R)LH-20, swelled in and eluated with MeOH. The product obtained ishomogenous according to TLC. Yield: 5.5 g (96%) Analysis: TLC in P₁(R_(f) : 0.28) [α]_(D) ²⁴ -33.0° (c 1.0, DMF)

III. Cbo-Pip-Arg(NO₂)- pNA

Performed according to II. Yield: 5.1 g (86%) Analysis: Homogenousaccording to TLC in P₁ (R_(f) : 0.30) [α]_(D) ²⁴ --26.2° (c 1.0, DMF)

IV. Cbo--Phe--Pip--Arg(NO₂)--pNA

2.9 g (5 mmol) Cbo--Pip--Arg(NO₂)--pNA are dicarbobenzoxylated in HBr inAcOH, precipitated and washed with ether and dried according to II.HBr--H--Pip--Arg(NO₂)--pNA is then dissolved in 15 ml of DMF. Thesolution is cooled to -10° C, made weakly basic with 0.9 ml of Et₃ N andfiltrated. 3.0 g (7.15 mmol) of Cbo--Phe--OpNP are added and then 0.65 g(5 mmol) of N-hydroxybenzotriazole as a catalyst. After 1 h further 0.35ml of Et₃ N is added and the same procedure repeated after 4 h. Thereaction solution is allowed to increase to room temperature over night.The solution is evaporated to dryness in a rotavapor. The residue isdissolved in EtOAc and treated with 2% sodium bicarbonate solution andwater and then evaporated. The residue is now dissolved in MeOH andchromatographed on Sephadex.sup.(R) LH-20 swelled and eluated with MeOH.The product obtained is homogenous according to TLC. Yield: 2.7 g (73%)Analysis: TLC in P₁ (R_(f) : 0.35) [α]_(D) ²⁴ -32.9° (c 1.0, DMF)

V. Cbo--D--Phe--Pip--Arg(NO₂)--pNA

Performed according to IV. Yield: 2.6 g (70%) Analysis: Homogenousaccording to TLC in P₁ (R_(f) : 0.44) [α]_(D) ²⁴ -38.4° (c 1.0, DMF)

VI. Cbo--Phe--Pro--Arg(NO₂)--pNA Performed according to IV. Yield: 2.9 g(80%) Analysis: Homegenous according to TLC in P₁ (R_(f) : 0.38) [α]_(D)²⁴ -39.2° (c 1.0, DMF)

VII. Cbo--D--Phe--Pro--Arg(NO₂)--pNA Performed according to IV. Yield:3.1 g (85%) Analysis: Homogenous according to TLC in P₁ (R_(fl) : 0.46)[α]_(D) ²⁴ -6.2° (c 1.0, DMF)

VIII. H--D--Phe--Pro--Arg--pNA.2HCl

175 mg (0.246 mmol) Cbo--D--Phe--Pro--Arg(NO₂)--pNA is deprotected byreaction with 5 ml of dry HF in the presence of 0.3 ml of anisole in anapparatus intended for this purpose according to Sakakibara for 60 minat 0° C. After finished reaction and after distillation of all HF theraw product is purified and submitted to ion exchange in two steps:

a. GPC on a column of Sephadex.sup.(R) G-15, swollen in 33% AcOH inwater, with the same medium as dissolving and eluation medium. The pureproduct is freeze dried from AcOH (aq).

b. Ion exchange on a column of Sephadex.sup.(R) QAE-25 in the chlorideform, swollen in MeOH:water (95:5) with the same medium as dissolvingand eluating medium. The pure product is freeze dried from water. Yield120 mg (80%) Analysis: Homogenous according to TLC in A (R_(f) : 0.29)Chloride content 11.53% (theoretically 11.6%) [α]_(D) ²⁴ -122° (c 0.5,50% AcOH (aq)

IX. H--Phe--Pro--Arg--pNA.2HCl

Performed according to VIII. Yield (71%) Analysis: Homogenous accordingto TLC in A (R_(f) : 0.22) Chloride content 11.0% (theoretically 11.6%)[α]_(D) ²⁴ -73.6° (c 0.5, 50% AcOH (aq)

X. H--D--Phe--Pip--Arg--pNA.2HCl

Performed according to VIII. Yield: (72%) Analysis: Homogenous accordingto TLC in A (R_(f) : 0.44) Chloride content 11.4% (theoretically 11.3%)[α]_(D) ²⁴ -109° (c 0.4, 50% AcOH (aq))

XI. H--Phe--Pip--Arg--pNA.2HCl Performed according to VIII. Yield: (55%)Analysis: Homogenous according to TLC in A (R_(f) : 0.41) Chloridecontent 11.3% (theoretically 11.3%) [α]_(D) ²⁴ -80.3° (c 0.5, 50% AcOH(aq))

XII. Cbo--D--Tyr(OBzl)--Pip--Arg(NO₂)--pNA Performed according to IV.Yield: 3.2 g (75%) Analysis: Homogenous according to TLC in P₁ (R_(f) :0.50)

XIII. H--D--Tyr--Pip--Arg--pNA

Performed according to VIII. Yield: (68%) Analysis: Homogenous accordingto TLC in P₁ (R_(f) : 0.44) Chloride content 10.8% (theoretically 11.1%)[α]_(D) ²⁴ -75.2° (c 0.5, 50% AcOH (aq))

XIV. Cbo--Aza--Arg(NO₂)--pNA Performed according to II. Yield: 4.2 g(75%) Analysis: Homegenous according to TLC in P₁ (R_(f) : 0.27)

XV. Cbo--D--Phe--Aze--Arg(NO₂)--pNA

Performed according to IV. Yield 2.4 g (69%) Analysis: Homogenousaccording to TLC in P₁ (R_(f) : 0.47)

XVI. H--D--Phe--Aze--Arg--pNA.sup.. 2HCl

Performed according to VIII. Yield: (71%) Analysis: Homogenous accordingto TLC in A (R_(f) : 0.21) Chloride content 11.6% (theoretically 11.9%)[α]_(D) ²⁴ -130° (c 0.5, 50% AcOH)

XVII. Cbo--Arg(NO₂)--βNA

7.2 g (20 mmol) of dry Cbo--Arg(NO₂)--OH are dissolved in 400 ml of THF.2.0 g (20 mmol) Et₃ N are added, whereupon the solution is cooled to-10° C under completely moisture free conditions. 2.7 g (20 mmol) ofisobutyl-chloroformate dissolved in 20 ml of THF are added to the cooledsolution during 10 min, and after another 10 min 344 g (20 mmol) ofβ-naphthylamine are added. The reaction mixture is allowed to each roomtemperature and is left at this temperature for 24 h. The reactionmixture is evaporated in vacuum to dryness, is treated 3-5 times withdistilled water, 3-5 times with a 5% sodium dicarbonate solution andagain 3-5 times with distilled water, after which it is dried in vacuum.The product is dissolved in MeoH and chromatographed on a column ofSephadex.sup.(R) LH-20, swelled in and eluated with MeOH. The productobtained is homogenous according to TLC in P₁. Yield: 8.1 g (84%)Analysis: Homogenous according to TLC in P₁ (R_(f) : 0.40) [α]_(D) ²³+7.4° (c 1.0, DMF)

XVIII. Cbo--Pip--Arg(NO₂)--αNA

Performed according to II. Yield: 4.8 g (82%) Analysis: Homogenousaccording to TLC in P₁ (R_(f) : 0.36)

XIX. Cbo--D--Phe--Pip--Arg(NO₂)--αNA

Performed according to IV. Yield: 2.6 g (71%) Analysis: Homogenousaccording to TLC in P₁ (R_(f) : 0.48)

XX. H--D--Phe--Pip--Arg--αNA.sup.. 2HCl

Performed according to VIII. Yield: (68%) Analysis: Homogenous accordingto TLC in A (R_(f) : 0.44) Chloride content 11.2% (theoretically 11.3%)[α]_(D) ²⁴ -105° (c 0.5, 50% AcOH (aq))

XXI. Cbo--Arg(NO₂)--αNA (4--OMe)

Performed according to XVII. Yield: 7.1 g (70%) Analysis: Homogenousaccording to TLC in P₁ (R_(f) : 0.42)

XXII. Cbo--Pip--ARG(NO₂)--αNA (4--OMe)

Performed according to II. Yield: 4.9 g (79%) Analysis: Homogenousaccording to TLC in P₁ (R_(f) : 0.38)

XXIII. Cbo--D--Phe--Pip--Arg(NO₂)--αNA (4--OMe)

Performed according to IV. Yield: 2.7 g (70%) Analysis: Homogenousaccording to TLC in P₁ (R_(f) : 0.51)

XXIV. H--D--Phe--Pip--Arg--αNA (4--OMe).sup.. 2HCl

Performed according to VIII. Yield: (64%) Analysis: Homogenous accordingto TLC in A (R_(f) : 0.44) Chloride content 10.4% (theoretically 10.7%)[α]_(D) ²⁴ -102° (c 0.5, 50% AcOH (aq))

Determination of thrombin by chromogenic substrates

The substrates prepared according to the examples are used fordetermination of thrombin as stated below.

The determnation principle is based upon the fact that the splittingproduct formed by enzymatic hydrolysis has a UV-spectrum which isessentially different from that of the substrate. Thus, the substrateaccording to Example X, H--D--Phe--Pip--Aet--pNA, has an absorptionmaximum at 315 nm and the molar extinction coefficient of 12500. At 405nm the absorption of the substrate has almost completely stopped.p-Nitroaniline, split off from the substrate during the enzymatichydrolysis, has an absorption maximum at 380 nm and a molar extinctioncoefficient of 13200 which at 405 nm only had decreased to 9620. Byspectrophotometrical determination at 405 nm it is thus possible toeasily follow the amount of p-nitroaniline formed, which is proportionalto the degree of the enzyamtic hydrolysis which in its turn isdetermined by the active amount of thrombin. For other substratesaccording to the invention rather identical conditions exist and forthis reason the determinations have consistently been made at 405 nm.

Table I shows a comparison of the relative reaction velocities betweenthe previously mentioned thrombin substrate S-2160, its non-benzoylatedform and substrate according to the invention. This table clearly showsthe superiority of the substrates according to the invention. They react4 times more rapidly with thrombin than the previously set substrate,S-2160, and are further about 10 times more soluble in water thanS-2160.

    ______________________________________                                                            Rel.                                                                          reaction  Solubility in                                   Substrate           velocity  H.sub.2 O (mg/ml)                               ______________________________________                                        A (S-2160)                                                                            Bz-Phe-Val-Arg-pNA                                                                            100       0.1                                         B       H-Phe-Val-Arg-pNA                                                                             3         1                                           IX      H-Phe-Pro-Arg-pNA                                                                             15        1                                           VIII    H-D-Phe-Pro-Arg-pNA                                                                           400       3                                           XI      H-Phe-Pip-Arg-pNA                                                                             9         1                                           X       H-D-Phe-Pip-Arg-pNA                                                                           420       3                                           ______________________________________                                    

Table I

Relative reactions velocity between thrombin (0.4 NIH/ml) and substrate(0.1 μmol/ml).

What is claimed is:
 1. Diagnostically active chromogenic substrate witha high specificity to thrombin and thrombin-like enzymes having thegeneral formula: ##STR3## and salts thereof, wherein R₁ is hydrogen orhydroxy, R₂ is nitrophenyl, and n is 1, 2 or
 3. 2. The substrate ofclaim 1 wherein R₁ is hydrogen.
 3. The substrate of claim 1 wherein R₁is hydroxy.
 4. The substrate of claim 1 wherein n is
 1. 5. The substrateof claim 1 wherein n is
 2. 6. The substrate of claim 1 wherein n is 3.7. The substrate of claim 1 which is H--D--Phe--Pro-Arg--P--NA or saltthereof.
 8. The substrate of claim 1 which is H--D--Phe--Pip--Art--pNAor salt thereof.
 9. The substrate of claim 1 which isH--D--Phe--Aze--Arg--pNA or salt thereof.
 10. The substrate of claim 1which is H--D--Tyr--Pip--Arg--pNA or salt thereof.